Monoclonal antibodies are the most widely used class of therapeutic proteins when high affinity and specificity for a target molecule are desired. However, non-antibody proteins having relatively defined three-dimensional structures that can be engineered to bind desired target molecules, commonly referred to as protein scaffolds, may have advantages over traditional antibodies due to their small size, lack of disulphide bonds, high stability, and ability to be expressed in prokaryotic hosts. These scaffolds typically contain one or more regions which are amenable to specific or random sequence variation, and such sequence randomization is often carried out to produce libraries of proteins from which desired products may be selected. Novel methods of purification are readily applied; scaffolds are easily conjugated to drugs/toxins, penetrate efficiently into tissues and can be formatted into multispecific binders (Binz and Pluckthun, Curr Opin Biotechnol, 16, 459-469, 2005; Skerra, J Mol Recognit, 13, 167-187, 2000).
One such protein scaffold is the fibronectin type III (FN3) domain identified in a multitude of proteins, having a characteristic tertiary structure with 6 loops connected by 7 beta strands. Three loops in particular, the FG, BC, and DE loops are structurally analogous to the complementarity determining regions (CDRs) of antibodies. These loops have been randomized to generate libraries of the FN3 domain scaffolds to successfully select specific binders to a number of different targets while retaining important biophysical properties (Getmanova et al., Chem Biol, 13, 549-556, 2006; Hackel et al., J Mol Biol, 381, 1238-1252, 2008; Karatan et al., Chem Biol, 11, 835-844, 2004; Koide et al., J Mol Biol, 284, 1141-1151, 1998; Koide et al., Proc Natl Acad Sci USA, 104, 6632-6637, 2007; Parker et al., Protein Eng Des Sel, 18, 435-444, 2005; Xu et al., Chemistry & Biology, 9, 933-942, 2002). Libraries of the FN3 domains have been generated by randomizing also the AB, EF and CD loops (U.S. Pat. Pub. No. 2011/0038866; Int. Pat. Pub. No. WO2011/05133; U.S. Pat. Pub. No. 2011/0124527). Other references for FN3 libraries include Int. Pat. Pub. Nos. WO2002/32925, WO2003/104418, WO2009/023184 and WO2010/060095. Int. Pat. Pub. No. WO2012/016245 describes FN3 domain libraries using CD and FG loops together with surface exposed residues of the beta-sheet.
It would be advantageous to obtain improved fibronectin domain scaffold proteins for both therapeutic and diagnostic purposes. The present disclosure provides such improved proteins.